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Laminin alpha1 chain synthesis in the mouse developing lung: requirement for epithelial-mesenchymal contact and possible role in bronchial smooth muscle development.

Schuger L, Skubitz AP, Zhang J, Sorokin L, He L - J. Cell Biol. (1997)

Bottom Line: In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains.Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin.Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from each other on their alpha chain, which is alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. Synthesis of laminin alpha1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

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ELISA showing different levels of LM α1 chain synthesis in different cocultures. (Column 1) Epithelial–mesenchymal  coculture established from mixed cell populations directly isolated from the lung. (Column 2) Epithelial–mesenchymal coculture established with cells from monocultures mixed in a 1:3 epithelial/mesenchymal ratio. (Column 3) Epithelial–mesenchymal  coculture established by adding epithelial cells to a mesenchymal  monolayer (both confluent at the time of determining LM-1 production). (Column 4) Epithelial–mesenchymal coculture established by adding mesenchymal cells to an epithelial monolayer  (both confluent at the time of determining LM-1 production).  The bars represent SD. The means and SD are based on quadruplicate examples in a single experiment. These were repeated  three times with similar results.
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Figure 3: ELISA showing different levels of LM α1 chain synthesis in different cocultures. (Column 1) Epithelial–mesenchymal coculture established from mixed cell populations directly isolated from the lung. (Column 2) Epithelial–mesenchymal coculture established with cells from monocultures mixed in a 1:3 epithelial/mesenchymal ratio. (Column 3) Epithelial–mesenchymal coculture established by adding epithelial cells to a mesenchymal monolayer (both confluent at the time of determining LM-1 production). (Column 4) Epithelial–mesenchymal coculture established by adding mesenchymal cells to an epithelial monolayer (both confluent at the time of determining LM-1 production). The bars represent SD. The means and SD are based on quadruplicate examples in a single experiment. These were repeated three times with similar results.

Mentions: Monocultures of lung epithelial and mesenchymal cells as well as homotypic cocultures (epithelial cells added to an established epithelial monoculture, E/E; or mesenchymal cells added to an established mesenchymal monoculture, M/M) synthesized LM α2, β1, and γ1 but not α1 chain (Fig. 2). Epithelial–mesenchymal cocultures synthesized LM α1 chain in addition to LM α2, β1, and γ1 chains (Fig. 2). There were, however, differences in the amount of LM α1 chain synthesized by the various heterotypic coculture systems. Epithelial–mesenchymal cocultures established by plating epithelial and mesenchymal cell populations directly after trypsinization of lungs (passage 0) synthesized the highest levels of LM α1 chain (Fig. 3, column 1), whereas cocultures established by adding one cell type on top of a heterotypic cell monolayer synthesized the lowest levels (Fig. 3, columns 3 and 4). Cocultures generated by plating a combination of epithelial and mesenchymal cells obtained from monocultures at a ratio of 1:3 to 1:5 synthesized intermediate levels of LM α1 chain (Fig. 3, column 2). In addition, these studies indicated that these cells may require at least 4 h of coculture for LM α1 chain to be synthesized, since LM α1 chains were not detected after 2 to 4 h of coculture by ELISA (not shown).


Laminin alpha1 chain synthesis in the mouse developing lung: requirement for epithelial-mesenchymal contact and possible role in bronchial smooth muscle development.

Schuger L, Skubitz AP, Zhang J, Sorokin L, He L - J. Cell Biol. (1997)

ELISA showing different levels of LM α1 chain synthesis in different cocultures. (Column 1) Epithelial–mesenchymal  coculture established from mixed cell populations directly isolated from the lung. (Column 2) Epithelial–mesenchymal coculture established with cells from monocultures mixed in a 1:3 epithelial/mesenchymal ratio. (Column 3) Epithelial–mesenchymal  coculture established by adding epithelial cells to a mesenchymal  monolayer (both confluent at the time of determining LM-1 production). (Column 4) Epithelial–mesenchymal coculture established by adding mesenchymal cells to an epithelial monolayer  (both confluent at the time of determining LM-1 production).  The bars represent SD. The means and SD are based on quadruplicate examples in a single experiment. These were repeated  three times with similar results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139794&req=5

Figure 3: ELISA showing different levels of LM α1 chain synthesis in different cocultures. (Column 1) Epithelial–mesenchymal coculture established from mixed cell populations directly isolated from the lung. (Column 2) Epithelial–mesenchymal coculture established with cells from monocultures mixed in a 1:3 epithelial/mesenchymal ratio. (Column 3) Epithelial–mesenchymal coculture established by adding epithelial cells to a mesenchymal monolayer (both confluent at the time of determining LM-1 production). (Column 4) Epithelial–mesenchymal coculture established by adding mesenchymal cells to an epithelial monolayer (both confluent at the time of determining LM-1 production). The bars represent SD. The means and SD are based on quadruplicate examples in a single experiment. These were repeated three times with similar results.
Mentions: Monocultures of lung epithelial and mesenchymal cells as well as homotypic cocultures (epithelial cells added to an established epithelial monoculture, E/E; or mesenchymal cells added to an established mesenchymal monoculture, M/M) synthesized LM α2, β1, and γ1 but not α1 chain (Fig. 2). Epithelial–mesenchymal cocultures synthesized LM α1 chain in addition to LM α2, β1, and γ1 chains (Fig. 2). There were, however, differences in the amount of LM α1 chain synthesized by the various heterotypic coculture systems. Epithelial–mesenchymal cocultures established by plating epithelial and mesenchymal cell populations directly after trypsinization of lungs (passage 0) synthesized the highest levels of LM α1 chain (Fig. 3, column 1), whereas cocultures established by adding one cell type on top of a heterotypic cell monolayer synthesized the lowest levels (Fig. 3, columns 3 and 4). Cocultures generated by plating a combination of epithelial and mesenchymal cells obtained from monocultures at a ratio of 1:3 to 1:5 synthesized intermediate levels of LM α1 chain (Fig. 3, column 2). In addition, these studies indicated that these cells may require at least 4 h of coculture for LM α1 chain to be synthesized, since LM α1 chains were not detected after 2 to 4 h of coculture by ELISA (not shown).

Bottom Line: In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains.Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin.Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from each other on their alpha chain, which is alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. Synthesis of laminin alpha1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

Show MeSH